丹参多糖的提取和结构表征、抗氧化活性评价
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摘要
丹参(Salvia miltiorrhiza Bunge)是唇形科(Lamiaceae)鼠尾草属(Salvia Linn.)多年生草本植物,以其根及根茎入药,它主要应用于治疗心血管疾病。目前国内外对丹参的开发利用主要集中在水溶性酚酸类和脂溶性二萜醌类等小分子化合物上,而对大分子物质多糖的研究尚处于起步阶段。本文以丹参为材料,分别采用热水浸提法、超声法、碱提法和酶法四种提取方法对其中的多糖进行了提取,并对这四种不同方法所提丹参多糖进行结构表征及抗氧化活性评价,以期为丹参多糖的开发利用提供科学依据。主要研究结果如下：
1.采用石油醚和乙醇回流脱脂,热水浸提法、超声法、碱提法和酶解法四种方法提取丹参中的多糖,提取液经Sevag试剂除蛋白,透析去除小分子物质,最后真空干燥得热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖。
2.热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的理化性质分析结果表明,热水浸提多糖和超声提取多糖的理化指标一致,与碱提多糖和酶提多糖之间差异较明显。
3.扫描电镜观察结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的形貌特征差别显著,超声提取法、碱提法和酶解法比传统的热水浸提法更有利于丹参多糖从细胞内溶出,它们的溶出效果顺序为：碱提多糖>超声提取多糖>酶提多糖>热水浸提多糖。
4.红外光谱分析结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖都具有多糖的一般特征吸收峰,红外光谱基本吻合,主要官能团没有显著差异,均为β-吡喃糖。另外,所提丹参多糖中存在结合蛋白和硫酸基。
5.氨基酸分析结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的氨基酸成分和含量有差别。碱提多糖含有16种必须氨基酸和非必须氨基酸(缬氨酸未检出),其余三种多糖均含有17种必须氨基酸和非必须氨基酸。热水浸提多糖、超声多糖、碱提多糖和酶提多糖的氨基酸含量分别为11.02%、16.87%、3.23%和4.85%,其中必须氨基酸分别为4.16%、6.71%、0.78%和1.60%。其中,超声提取多糖在氨基酸含量和必须氨基酸含量两个指标均处于最高,碱提多糖两个指标最低。
6.抗氧化活性评价表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖均有一定的抗氧化活性。在4.0 mg/mL的样品浓度下,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖对DPPH自由基清除率的最大值分别为84.09%、83.73%、37.34%和82.23%；在0.40 mg/mL的样品浓度下,对超氧阴离子自由基清除率的最大值分别为86.72%、89.45%、76.80%和81.91%；在0.40 mg/mL的样品浓度下,对羟基自由基清除率最大值分别为42.89%、93.58%、25.20%和47.90%。
As a famous traditional chinese medicine, DanShen(Salvia miltiorrhiza Bunge) is a perennial herb which comes from Salvia, the largest genus of the Lamiaceae family. The roots and rhizomes have been used as herbal medicine for the treatment of cadiovascular disease for several thousands of years in China. Currently, The exploitation of DanShen is mainly focused on micromolecules such as soluble phenolic acids and fat-soluble diterpene quinone, while the macromolecules such as polysaccharides are still in its infancy. In this work, the polysaccharides from Danshen were extracted by hot water, ultrasonic, alkali and enzyme respectively. The structure of polysaccharides from Danshen were analyzed by several methods such as physicochemical properties analysis, SEM, IR and amino acid analysis. The antioxidant activities were examined by several methods, such as the DPPH radical scavenging assay, superoxide radical scavenging assay, hydroxyl radical scavenging assay. The purpose is to provide the scientific basis for effective development. The main research results are as follows:
1. Danshen was defatted with petroleum ether and ethanol, then defatted extraction were execution by hot water, ultrasonic, alkali and enzyme respectively. Then they were subjected to Sevag method to remove free protein and to dialyse in order to remove micromolecules. Finally, four polysaccharides from Danshen were obtained by vacuum drying.
2. The results of physicochemical properties analysis in four kinds of polysccharides from DanShen showed that the index of polysccharides by hot water and ultrasonic were exactly the same, while significantly different among polysccharides by alkali and enzyme.
3. The results of SEM showed that the surface features of different polysccharides had significantly difference. They can easily be identified. Their dissolution efficiency were as follows:polysaccharide by alkali>polysaccharide by ultrasonic> polysaccharide by enzyme> polysaccharide by hot water.
4. The results of IR analysis showed that four kinds of polysccharides had the general characteristic absorption peak of polysaccharides. The major functional groups were no significant different. Futhermore, they were allβ-pyranose. Then the results showed that they all have binding proteins and sulfate groups.
5. The results of amino acid analysis showed that the composition and contents of amino acid in four kinds of polysccharides have significantly difference. The polysccharide by alkali have 16 kinds of essential amino acid and non-essential amino acid (Val is not detected). The others have 17 kinds of essential amino acid and non-essential amino acid. The amino acid contents of the four kinds of polysccharides were 11.04%, 16.88%,3.23% and 4.85% respectively. The essential amino acid contents of the four kinds of polysccharides were 4.17%,6.72%,0.78% and 1.59% respectively. It was worth nothing that the quata of the polysccharide by ultrasonic in the amino acid contents and the essential contents were the highest,16.88% and 6.72% respectively. On the contrary, the index of the polysccharides by alkali was the lowest.
6. The results of evaluation of antioxidant activity showed that four kinds of polysccharides have some antioxidant activity. In the 0.40 mg/mL concentration of sample, the maximum value of DPPH radical scavenging were 84.09%,83.73%,37.34% and 82.23% respectively. In the 0.40 mg/mL concentration of sample, the maximum value of superoxide radical scavenging were 86.72%,89.45%,76.80% and 81.91% respectively. In the 0.40 mg/mL concentration of sample, the maximum value of hydroxyl radical scavenging were 42.89%,93.58%,25.20% and 47.90% respectively.
1.采用石油醚和乙醇回流脱脂,热水浸提法、超声法、碱提法和酶解法四种方法提取丹参中的多糖,提取液经Sevag试剂除蛋白,透析去除小分子物质,最后真空干燥得热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖。
2.热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的理化性质分析结果表明,热水浸提多糖和超声提取多糖的理化指标一致,与碱提多糖和酶提多糖之间差异较明显。
3.扫描电镜观察结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的形貌特征差别显著,超声提取法、碱提法和酶解法比传统的热水浸提法更有利于丹参多糖从细胞内溶出,它们的溶出效果顺序为：碱提多糖>超声提取多糖>酶提多糖>热水浸提多糖。
4.红外光谱分析结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖都具有多糖的一般特征吸收峰,红外光谱基本吻合,主要官能团没有显著差异,均为β-吡喃糖。另外,所提丹参多糖中存在结合蛋白和硫酸基。
5.氨基酸分析结果表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖的氨基酸成分和含量有差别。碱提多糖含有16种必须氨基酸和非必须氨基酸(缬氨酸未检出),其余三种多糖均含有17种必须氨基酸和非必须氨基酸。热水浸提多糖、超声多糖、碱提多糖和酶提多糖的氨基酸含量分别为11.02%、16.87%、3.23%和4.85%,其中必须氨基酸分别为4.16%、6.71%、0.78%和1.60%。其中,超声提取多糖在氨基酸含量和必须氨基酸含量两个指标均处于最高,碱提多糖两个指标最低。
6.抗氧化活性评价表明,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖均有一定的抗氧化活性。在4.0 mg/mL的样品浓度下,热水浸提多糖、超声提取多糖、碱提多糖和酶提多糖对DPPH自由基清除率的最大值分别为84.09%、83.73%、37.34%和82.23%；在0.40 mg/mL的样品浓度下,对超氧阴离子自由基清除率的最大值分别为86.72%、89.45%、76.80%和81.91%；在0.40 mg/mL的样品浓度下,对羟基自由基清除率最大值分别为42.89%、93.58%、25.20%和47.90%。
As a famous traditional chinese medicine, DanShen(Salvia miltiorrhiza Bunge) is a perennial herb which comes from Salvia, the largest genus of the Lamiaceae family. The roots and rhizomes have been used as herbal medicine for the treatment of cadiovascular disease for several thousands of years in China. Currently, The exploitation of DanShen is mainly focused on micromolecules such as soluble phenolic acids and fat-soluble diterpene quinone, while the macromolecules such as polysaccharides are still in its infancy. In this work, the polysaccharides from Danshen were extracted by hot water, ultrasonic, alkali and enzyme respectively. The structure of polysaccharides from Danshen were analyzed by several methods such as physicochemical properties analysis, SEM, IR and amino acid analysis. The antioxidant activities were examined by several methods, such as the DPPH radical scavenging assay, superoxide radical scavenging assay, hydroxyl radical scavenging assay. The purpose is to provide the scientific basis for effective development. The main research results are as follows:
1. Danshen was defatted with petroleum ether and ethanol, then defatted extraction were execution by hot water, ultrasonic, alkali and enzyme respectively. Then they were subjected to Sevag method to remove free protein and to dialyse in order to remove micromolecules. Finally, four polysaccharides from Danshen were obtained by vacuum drying.
2. The results of physicochemical properties analysis in four kinds of polysccharides from DanShen showed that the index of polysccharides by hot water and ultrasonic were exactly the same, while significantly different among polysccharides by alkali and enzyme.
3. The results of SEM showed that the surface features of different polysccharides had significantly difference. They can easily be identified. Their dissolution efficiency were as follows:polysaccharide by alkali>polysaccharide by ultrasonic> polysaccharide by enzyme> polysaccharide by hot water.
4. The results of IR analysis showed that four kinds of polysccharides had the general characteristic absorption peak of polysaccharides. The major functional groups were no significant different. Futhermore, they were allβ-pyranose. Then the results showed that they all have binding proteins and sulfate groups.
5. The results of amino acid analysis showed that the composition and contents of amino acid in four kinds of polysccharides have significantly difference. The polysccharide by alkali have 16 kinds of essential amino acid and non-essential amino acid (Val is not detected). The others have 17 kinds of essential amino acid and non-essential amino acid. The amino acid contents of the four kinds of polysccharides were 11.04%, 16.88%,3.23% and 4.85% respectively. The essential amino acid contents of the four kinds of polysccharides were 4.17%,6.72%,0.78% and 1.59% respectively. It was worth nothing that the quata of the polysccharide by ultrasonic in the amino acid contents and the essential contents were the highest,16.88% and 6.72% respectively. On the contrary, the index of the polysccharides by alkali was the lowest.
6. The results of evaluation of antioxidant activity showed that four kinds of polysccharides have some antioxidant activity. In the 0.40 mg/mL concentration of sample, the maximum value of DPPH radical scavenging were 84.09%,83.73%,37.34% and 82.23% respectively. In the 0.40 mg/mL concentration of sample, the maximum value of superoxide radical scavenging were 86.72%,89.45%,76.80% and 81.91% respectively. In the 0.40 mg/mL concentration of sample, the maximum value of hydroxyl radical scavenging were 42.89%,93.58%,25.20% and 47.90% respectively.
引文
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